Project Summary: Molecular reagents that can site-specifically target loci have transformed genome engineering in animals. In particular, RNA-programmable gene targeting nucleases like Cas9 can create double stranded breaks at defined loci, followed by homology directed repair with a donor nucleic acid molecule containing homology on either side of the double stranded break. However, most efforts to date have focused on gene replacement for relatively short stretches of nucleic acids. This is because site-specific insertion/replacement of multi-kilobases long DNA fragments into mammalian genomes is technically challenging. Solving this grand challenge is important for a number of fields that span discovery biology and biotechnology: (i.) generation of animal models including conditional knockout mice and mice with inducible, reporters, recombinases, transcriptional activators and transgenic models expressing long protein coding and multi-cistronic cassettes; (ii.) high efficiency insertion of transgenes in primary cell lines at site-specific locations; (iii.) development of diverse antibody libraries using mammalian cells; and (iv.) developing programmable, genetically engineered T cells for immunotherapy To address the long-term goal of highly efficient site-specific addition of multi-kb DNA cassettes into animal genomes, the applicants have recently published a partial solution called Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR). The method has been used at over a dozen loci revealing robustness, high efficiency and, moreover, versatility as it can create conditional as well as knock-in alleles. The key to the high efficiency of DNA cassette insertion of the Easi-CRISPR method is the use of a long ssDNA donor molecule, but current methods to prepare multi-kb linear ssDNA are insufficient for 2-10 kb inserts. Thus, the primary objective of this project is to develop a robust and simple method for preparation of multi-kb linear ssDNA donors and to validate long ssDNA donors with Easi-CRISPR for genome engineering in animals. The rationale for the proposed research is guided by the urgent need for facile methods of animal genome engineering in complex disease models and supported by the applicants? preliminary data on ssDNA preparation and utility of Easi- CRISPR The proposed research project will be carried out by pursuing two specific aims: 1) Optimize method for preparation of long ssDNA; 2) Test long ssDNA for generation of transgenic animals using the Easi-CRISPR approach This approach is innovative because it combines the key insights from different fields of biomolecular engineering and mouse genetics in unique ways, and it is significant in providing a generalized method for preparing long, linear ssDNA that can be broadly used by the worldwide research community for genome engineering of large inserts at site-specific locations.